Project Summary HIV Nef downregulates cell surface host proteins, including MHC-I and SERINCs, by redirecting them into clathrin coated vesicles (CCVs) for lysosomal degradation. Nef achieves this by hijacking the AP-1 and AP-2 clathrin adaptors. HIV-1 Nef cooperates with the GTPase Arf1 to induce oligomerization and activation of AP-1, assembling as either dimers or closed or open trimers in vitro. In aim 1, we will test the new hypothesis that Nef is a cargo-dependent allosteric switch that regulates the conformational state of the AP-1:Arf1 trimer. In this model, Nef is able, depending on the type of cargo bound, to induce AP-1:Arf trimers to adopt an open state that promotes CCV formation, or to form a closed trimer state that retains cargo at the trans-Golgi network (TGN). The best characterized substrates for Nef at the TGN are MHC-I (all Nefs) and tetherin (SIV, HIV-2, and group O HIV-1 Nefs). A substantial body of data suggest that tetherin accumulates at the TGN, while MHC-I is degraded in the lysosome. We found that tetherin and MHC-I promote the closed and open conformations of AP-1 trimers, respectively. Here, I will identify the structural determinants that control the equilibrium between the three known oligomeric states of AP-1 (dimer, closed trimer, and open trimer). Aim 2 will investigate how a poorly understood class of transmembrane proteins, the SERINCs, are downregulated by Nef. This question is of paramount importance in the HIV field because the SERINCs are the main target of Nef's infectivity-enhancing activity. SERINCs lack canonical AP internalization signals and there has been no substantial evidence for direct SERINC-Nef binding. Here, we hypothesize that a novel AP-2 binding site might be created by phosphorylation of SERINC cytosolic loops. We provide preliminary evidence in support of this concept, and plan to use this as the starting point to determine the structural basis for SERINC downregulation. In our current working model for Nef and AP-2 downregulation of SERINCs, the Nef LL sequence promotes AP-2 unlocking, while a non-canonical phosphorylated sequence in one of the cytosolic loops of SERINC3 binds to the ?2 CTD domain. Aim 2 will provide a direct visualization of this key event by cryo-EM.